Nucleotide and protein sequences of nogo genes and methods based thereon

ABSTRACT

The present invention relates to the gene, $i(Nogo), its encoded protein products, as well as derivatives and analogs thereof. Production of Nogo proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.

This application is a continuation application of U.S. Ser. No. 12/290,316 filed Oct. 29, 2008 which is a divisional application of U.S. Ser. No. 09/830,972, filed Sep. 24, 2001, which is a national stage entry of PCT application No. PCT/US99/26160, filed Nov. 5, 1999, which claims the benefit of U.S. provisional application 60/107,446, filed Nov. 6, 1998. Each of these applications are herein incorporated by reference in their entirety.

1. INTRODUCTION

The present invention relates to the gene, Nogo, and in particular to Nogo, its encoded protein products, as well as derivatives and analogs thereof. Production of Nogo proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.

2. BACKGROUND OF THE INVENTION

In the central nervous system (CNS) of higher vertebrates, regeneration of axons after injury is almost absent and structural plasticity is limited. Growth inhibitors associated with CNS myelin are likely to play an important role. This is evidenced by a monoclonal antibody (mAb), IN-1, that neutralizes a potent neurite growth inhibitory myelin protein, thereby promoting long-distance axonal regeneration and enhancing compensatory plasticity following spinal cord or brain lesions in adult rats.

A number of in vitro and in vivo observations have revealed a new aspect of neurite growth regulation which is the presence of repulsive and inhibitory signals and factors (Keynes and Cook, 1995, Curr. Opin. Neurosci. 5: 75-82). Most of these signals seemed to be proteins or glycoproteins. A first breakthrough towards identification of the factors was the purification and cDNA cloning of a chick brain-derived growth cone collapse inducing molecule, Collapsin-1, now called Semaphorin 3A.

A second group of repulsive guidance cues recently purified and cloned is now designated as Ephrins. They are ligands for the Eph receptor tyrosine kinase family.

Ephrin-A5 and Ephrin-A2 are expressed as gradients in the optic tectum of the chick embryo, and their ectopic expression or deletion causes guidance errors of ingrowing retinal axons. Like the Semaphorins, the Ephrin family has 15 to 20 members, each with a complex and dynamic expression in and outside of the nervous system. The functions of most of these molecules remain to be analyzed.

A third group of guidance cues which can repulse growing axons and is expressed in the developing nervous system are the Netrins. Netrin has been purified as a floor plate derived chemoattractant for commissural axons in early spinal cords, like its C. elegans ortholog unc-6. Netrin-1 turned out to have repulsive effects for certain types of neurons-depending on the type of receptor present on the target neuronal growth cones (Tessier-Lavigne et al., 1996, Science 274: 1123-33).

Previously, a potent neurite growth inhibitory activity associated with adult CNS oligodendrocytes and myelin was reported by Canoni and Schwab (1988, J. Cell Biol. 106: 1281-1288). A major constituent is a high molecular weight membrane protein (NI-250, with a smaller component, NI-35, in rat) which was recently purified, and which is related to the subject of the present invention, and is bound by the neutralizing mAb, IN-1 (Canoni and Schwab, 1988, J. Cell Biol. 106: 1281-1288; U.S. Pat. Nos. 5,684,133; 5,250,414; PCT Publication WO 93/00427).

Myelin-associated neurite growth inhibitors play a crucial role in preventing regeneration of lesioned CNS axons. When oligodendrocyte development and myelin formation is blocked in chicken or rats, the regeneration permissive period following CNS lesions is prolonged. Indeed, myelin formation coincides in time with the end of the developmental period where the CNS shows high structural plasticity and a high potential for regeneration.

NI-250 and NI-35 are likely to be major components of the myelin-associated growth inhibition as evidenced by in vivo application of IN-1 to spinal cord lesioned adult rats which induced regeneration of corticospinal axons over long distances and allowed motor and behavior functional recovery especially with regard to locomotion. Similar experiments on the optic nerve and the cholinergic septo-hippocampal pathway also demonstrated the in vivo relevance of the IN-1 recognized antigen, NI-35/250 (Schnell and Schwab, 1990, Nature 343: 269-272; Bregman et al., 1995, Nature 378: 498-501).

Unlesioned fiber systems also respond to the neutralization of neurite growth inhibitors by IN-1. Recent experiments have conclusively shown that following a selective corticospinal tract lesion (pyramidotomy), intact fibers sprout across the midline in the spinal cord and brainstem and establish a bilateral innervation pattern, accompanied by an almost full behavioral recovery of precision movements in the presence of IN-1 (Z'Graggen et al., 1998, J. Neuroscience 18 (12): 4744-4757).

Isolation of the gene that encodes the neurite growth inhibitory protein provides multiple opportunities for developing products useful in neuronal regeneration and in treatment of various neurological disorders, including CNS tumor.

Citation of a reference hereinabove shall not be construed as an admission that such reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of Nogo genes (human, rat and bovine Nogo and Nogo homologs of other species), and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided.

In a specific embodiment, the Nogo protein is a rat, bovine or human protein.

The invention also relates to a method of identifying genes which interact with Nogo.

Nogo is a gene provided by the present invention, identified by the method of the invention, that both encodes and interacts with neural growth regulatory proteins.

The invention also relates to Nogo derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a naturally occurring Nogo protein. For example, a major inhibitory region between amino acids 542 to 722 have been identified. Such functional activities include, but are not limited to, neurite growth inhibition of neural cells, spreading and migration of fibroblasts, or any cell exhibiting neoplastic growth, the ability to interact with or compete for interaction with neural growth regulatory proteins, antigenicity which is the ability to bind (or compete with Nogo for binding) to an anti-Nogo antibody, immunogenicity which is the ability to generate antibody which binds to Nogo. These antibodies having the potential to induce neurite outgrowth or prevent dorsal root ganglia growth cone collapse by inhibiting the function of Nogo, and functional fragments or derivatives of Nogo, with the ability to inhibit neurite outgrowth.

The invention further relates to fragments (and derivatives and analogs thereof) of Nogo which comprise one or more domains of a Nogo protein such as the acidic and proline rich amino terminus (e.g., at amino acids 31 to 58 of SEQ ID NO: 2), the highly conserved carboxy terminus, and two hydrophobic stretches of 35 and 36 amino acids length in rat Nogo, also in the carboxy terminus (e.g., at amino acids 988 to 1023, and at 1090 to 1125 of SEQ ID NO: 2).

Antibodies to the various Nogo, and Nogo derivatives and analogs, are additionally provided. In particular, by way of example, two antibodies have been derived, the first antibody, termed AS 472, was derived using as immunogen a synthetic peptide corresponding to amino acids 623 to 640 of SEQ ID NO: 2, and the second antibody, termed AS Bruna, was generated against the carboxy-terminus, amino acids 762 to 1163 of SEQ ID NO: 2, of Nogo.

Methods of production of the Nogo proteins, derivatives and analogs, e.g., by recombinant means, are also provided.

The present invention also relates to therapeutic and diagnostic methods and compositions based on Nogo proteins and nucleic acids. Therapeutic compounds of the invention include but are not limited to Nogo proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the Nogo proteins, analogs, or derivatives; and Nogo ribozymes or Nogo antisense nucleic acids.

The present invention also relates to therapeutic and diagnostic methods and compositions based on Nogo proteins and nucleic acids and anti-Nogo antibodies. The invention provides for treatment of CNS and neural derived tumors by administering compounds that promote Nogo activity (e.g., Nogo proteins and functionally active analogs and derivatives including fragments thereof; nucleic acids encoding the Nogo proteins, analogs, or derivatives, agonists of Nogo).

The invention also provides for treatment of diseases, disorders or damage which ultimately result in damage of the nervous system; such diseases, disorders or damage include, but are not limited to, central nervous system (CNS) trauma, (e.g. spinal cord or brain injuries), infarction, infection, malignancy, exposure to toxic agents, nutritional deficiency, paraneoplastic syndromes, and degenerative nerve diseases (including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's Chorea, multiple sclerosis, amyotrophic lateral sclerosis, and progressive supra-nuclear palsy); by administering compounds that interfere with Nogo activity (e.g., a dominant negative Nogo derivative; antibodies to Nogo; anti-sense nucleic acids of Nogo; Nogo ribozymes or chemical groups that bind an active site of Nogo).

Animal models, diagnostic methods and screening methods for predisposition to disorders, and methods to identify and evaluate Nogo agonists and antagonists, are also provided by the invention.

3.1 DEFINITIONS

As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, “Nogo” shall mean the Nogo gene, whereas “Nogo” shall indicate the protein product of the Nogo gene.

4. DESCRIPTION OF THE FIGURES

FIG. 1 a-1 b: (a) Nogo cDNA clones: CWP1-3 is a bovine cDNA clone isolated from the screening of a bovine spinal cord white matter cDNA library with degenerated oligonucleotides MSC5-8 (pooled) and MSC9. Complementary RNA derived from this clone was used for subsequent rat cDNA library screening. O1i3 and 01118 are isolated from an oligo d (T)-primed rat oligodendrocyte library. R1-3U21, R018U1 and R018U37-3 are isolated from a hexanucleotides-primed rat brain stem/spinal cord library (Stratagene).

The positions of the 6 bovine NI220 (bNI220) peptide sequences are indicated on CWP1-3 and R13U21. Sequences at the junctions of different exons are marked on top of each clone. The question marks indicated on R018U1 identify the sequence on this clone which does not match sequences from any other Nogo clones. RO18U37-3 was sequenced-only from the 5′-end, and the unsequenced portion is represented by dots. (b) Schematics demonstrating the hypothetical mechanism for the generation of three Nogo transcripts. PI and P2 represent the putative location of the alternative promoters. The minimum number of three exons is required for generating the three transcripts as shown schematically, although each exon could potentially be split into multiple exons.

FIG. 2 a-2 b: (a) Nucleotide (SEQ ID NO: 1) and amino acid sequences (SEQ ID NO: 2) of Nogo transcript A (sequence generated by connecting RO18U37-3, O1i18, and R1-3U21 cDNA sequences). Oval box: presumed initiation codon; underlined with dots: acidic stretch; D: potential PKC sites; A: potential casein kinase II sites; thick underline: carboxy terminal hydrophobic regions and potential transmembrane domains; thin underline: potential N-glycosylation sites. (b) Peptide sequence comparison between sequenced, purified bovine NI220 (bNI220; SEQ ID NOS: 3-8), and the corresponding bovine (SEQ ID NOS: 9-14) and rat (SEQ ID NOS: 15-20) sequences translated from rat and bovine cDNAs.

Rat and bovine amino acid sequences, which do not match the bNI220 peptide sequences, are in lower case.

FIG. 3 a-3 b: (a) Amino acid sequence comparison of the carboxy terminal 180 amino acid common regions of NSP (human; SEQ ID NO: 21), S-REX (rat) (SEQ ID NO: 22), CHS-REX (chicken; SEQ ID NO: 23), NOGOBOV (bovine; SEQ ID NO: 24), NOGORAT (rat; SEQ ID NO: 25), a C. elegans EST clone (WO6A7A; SEQ ID NO: 26), and a D. melanogaster EST clone (US51048; SEQ ID NO: 27). (b) Evolutionary conservation of the two hydrophobic regions. Percent similarities within and across species of the common hydrophobic regions are shown. Shaded letters: conserved amino acids.

FIG. 4 a-4 c: (a) Northern hybridization of various tissues with the Nogo common probe. The common probe contains transcript A sequence between nucleotides 2583-4678.

ON, optic nerve; SC, spinal cord; C, cerebral cortex; DRG, dorsal root ganglia; SN, sciatic nerve; PC 12, PC 12 cell line. (b) Northern hybridization of spinal cord and PC 12 cells RNAs with an exon 1-specific probe (left panel) and of hindbrain (IIB) and skeletal muscle (M) RNAs with an exon 2 specific probe (right panel). (c) Northern hybridization with the Nogo common probe. K, kidney; B, cartilage (from breastbone); Sk, skin; M, skeletal muscle; Lu, lung; Li, liver; Sp, spleen. The three major transcripts are marked (4.6 kilobases (kb), 2.6 kb, and 1.7 kb). A: a diffuse but consistent band about 1.3 kb in size.

FIG. 5 a-5 f: In situ hybridization of adult rat spinal cord and cerebellum sections.

(a, d) Rows of oligodendrocytes (OL) in spinal cord and cerebellum white matter, respectively, can be seen labeled by the Nogo antisense “common” riboprobe. This is very similar to the signals detected when a consecutive spinal cord section was hybridized to an antisense plop riboprobe (b). (c) Neurons in grey matter (GM) were also labeled by the Nogo antisense “common” riboprobe. WM: white matter. Bright field and fluorescent view, respectively, of a cerebellum section double labeled with the Nogo antisense “common” riboprobe (e) and of an anti-GFAP antibody (f). Purkinje cells (double arrowheads) are strongly labeled with the Nogo probe, while astrocytes (arrowheads, black and white) are negative. A few cells in the granular cell layer (Gr) are also labeled with the Nogo probe, m: molecular layer. Scale bar: a, b, d-f: 50 p. m; c: 280 p. m.

FIG. 6 a-6 i: In situ hybridization of optic nerves at different postnatal days (a, f: PO; b, g: P3; c, h: P7; d, e, is P22) with either Nogo orplp (antisense or sense) probes. (a-d) Nogo antisense probe; (e) Nogo sense probe; (g-i) plp antisense probe; (f) plp sense probe.

Nogo expression in oligodendrocyte precusors can be detected as early as PO, while plp expression was only beginning to be detectable in P3 optic nerves close to the chiasm (g).

FIG. 7: AS Bruna and AS 472 both recognize a myelin protein of about 200 kd).

Rat myelin extract and bovine q-pool were prepared according to Spillmann et al, 1998, J. Biol. Chem., 273 (30): 19283-19293. AS Bruna and AS 472 each recognized a 200 kD band as well as several lower bands in bovine myelin, which may be breakdown products of bNI220. AS Bruna stained a band 200 kD in rat myelin. I: AS Bruna; P: AS Bruna, preimmune serum; E: AS 472 affinity purified.

FIG. 8 a-8 i: Immunohistochemistry on rat spinal cord and cerebellum using IN-1 (a-e), AS Bruna (d-f), and AS 472 (g-i), as indicated at the left of each panel. A strong myelin staining was observed in both tissues with all three antibodies when the frozen sections were fixed with ethanol/acetic acid (a, b, d, e, g, h). Treatment of the sections with methanol abolished the myelin staining except for oligodendrocyte cell bodies (arrows; c, f, i). Arrowheads: Purkinje cells, WM, white matter; GM, grey matter, DR: dorsal root; Gr, granular cell layer; m, molecular layer. Scale bar: a, d, g: 415Zm; b, c, e, f, h, is 143m.—FIG. 9 a-9 d: Neutralizing activity of AS 472 and AS Bruna in different bioassays.

(a) the NIH 3T3 fibroblasts were plated on cell culture dishes coated with q-pool and pre-treated with IN-1, AS Bruna, AS 472 or the corresponding pre-immune sera. Both polyclonal sera showed even a slightly better neutralization of the inhibitory substrate than IN-1. The pre-immune sera had no significant effect on the spreading of the NIH 3T3 cells.

Addition of an excess of the peptide (P472) that was used to raise AS 472 competed the neutralizing activity whereas an unspecific peptide (Px) had no effect. (b) Pre-treatment of the inhibitory substrate with AS Bruna or AS 472 resulted in DRG neurite outgrowth comparable to what can be observed on a laminin substrate. Examples for neurite outgrowth from DRG on q-pool pre-treated with PBS (c; score=0) and pretreated with AS Bruna (d; score=4).

FIG. 10 a-10 d: Injection of optic nerve explants with AS 472 results in ingrowth of axons. (a) Pairs of adult rat optic nerves were dissected, injected with AS 472 or preimmune serum and placed into chamber cultures such that one end of the nerves was in contact with dissociated PO rat DRG neurons. (b) After two weeks in vitro, EM sections of the nerves were taken at 3.5 mm from the cut site (arrows in A) and systematically screened for intact axons (3 experiments). (c) Regenerated axon bundles (arrows) grow through degenerating AS 472 injected optic nerve. (d) Regenerating axons in contact with myelin.

Magnification: c, d, 35,000×.

FIG. 11 a-11 c: Recombinant Nogo A expression in transfected COS cells. (a) Western blot showing immunoreactivity of AS Bruna to recombinant Nogo A (lane 2) and endogenous Nogo A from primary cultured rat oligodendrocytes (lane 3). The mobilities of these two proteins are virtually identical at about 200 kD on a 5% denaturing SDS gel. A control LacZ construct transfected sample (lane 1) showed no immunoreactivity with AS Bruna. The same blot was also probed with anti-myc antibody, 9E10, as indicated. The band that reacted with AS Bruna also reacted with the anti-myc tag antibody, 9E10 (lane 5), while the endogenous Nogo A did not (lane 6). The LacZ control transfection sample showed the expected band at about 118 kD (lane 4). COS cells transiently transfected with a Nogo A construct were double stained with AS Bruna (b) and IN-1 (c). Cells positively stained with AS Bruna were also positive with IN-1.

FIG. 12: The nucleotide sequence (SEQ ID NO: 28) of the bovine Nogo cDNA.

FIG. 13: The amino acid sequence of rat Nogo A (SEQ ID NO: 2) aligned with the theoretical amino acid sequence of human Nogo (SEQ ID NO: 30). The human Nogo amino acid sequence was derived from aligning expressed sequence tags (EST) to the rat Nogo sequence and translating the aligned human ESTs using the rat Nogo as a guiding template.

FIG. 14: Rat Nogo C nucleic acid (SEQ ID NO: 31) sequence and its corresponding amino acid sequence (SEQ ID NO: 32).

FIG. 15 a-15 e: Nogo A is present on the oligodendrocyte plasma membrane, as demonstrated by immunocytochemistry and cell surface biotinylation of oligodendrocytes in culture.

Immunocytochemistry (a-d): Oligodendrocytes from optic nerves of P10 rats were dissociated and cultured for 2 days. Staining of live cells with mAb IN-1 (a) or AS 472 (c) showed immunoreactivity on oligodendrocyte cell bodies and processes. In the presence of the competing peptide P472, AS 472 showed only background labeling (all cell types) (d).

Similar non-specific staining was seen when primary antibodies were omitted (b).

Evaluation: Number-coded dishes were randomly mixed and classified by 3 independent observers. 8/10 dishes were correctly classified AS 472-positive, mAb IN-1-positive or controls by all three observers.

Biotinylation (e): Rat P4 whole brain cultures enriched in oligodendrocytes were cell surface biotinylated with a membrane impermeable reagent after seven days in culture.

Subsequently, cell homogenates were treated with streptavidin-Dynabeads. Precipitate (Ppt) and supernatant (sup) were blotted with AS472; they showed a distinct protein pattern: Cell surface Nogo A found in the precipitate showed a higher apparent molecular weight than intracellular Nogo A. This shift is probably due to glycosylation. The luminal ER protein BiP and the large majority of p-tubulin could only be found in the intracellular fraction.

FIG. 16 a-16 j: Functional assays show the presence of Nogo A on the cell membrane of oligodendrocytes. Pre-incubation of optic nerve cultures with AS 472 (a, b) allowed the NIH 3T3 fibroblasts to spread over the highly branched oligodendrocytes which are outlined by immunofluorescent staining for GalC (01 antibody) (a). Arrows in the corresponding phase contrast image (b) indicate the NIH 3T3 fibroblasts spreading on top of the oligodendrocytes. (c, d): When AS 472 was added together with P472, the NIH 3T3 fibroblasts strictly avoided the territories of the GaIC-positive oligodendrocytes (arrowheads) (Caroni and Schwab, 1988 Neuron 1: 85-96). (e, f): In the presence of AS 472, PO rat dissociated DRG neurons were able to extend neurites over the territory of highly branched oligodendrocytes (arrows in f). (g, h): The peptide P472 efficiently competed the neutralizing activity of AS 472: the neurites completely avoided the oligodendrocytes. AS 472 used in these experiments was generated against the rat 472 peptide sequence. (i, j): Quantification of these results (as described in methods) demonstrated the strong neutralizing activity of AS 472 in both types of assays. Scale bar: 40 llm.

FIG. 17 a-17 e: Recombinant Nogo A is an inhibitory substrate and its inhibitory activity is neutralized by mAb IN-1. RecNogo A enriched extracts from a stable CHO-Nogo A cell line, or P-galactosidase, isolated in parallel from the stable CHO-LacZ cell line, were coated for the NTH 3T3 fibroblast spreading and DRG neurite outgrowth assays.

(a) Silver gel of myc-his-tagged recLacZ (lane 1) and recNogo A (lane 2) shows the Nogo A band at 180 kD. The identity of the Nogo A band was confirmed by Western blot incubated with AS Bruna (lane 3) and an anti-myc antibody 9E10 (lane 4). (b) RecNogo A coated dishes were clearly inhibitory to the NIH 3T3 spreading. Pre-incubation with mAb IN-1 or AS Bruna resulted in a highly significant (p<0.01) neutralization of inhibitory activity. The control 1 μM mAb O 1 and pre-immune serum were ineffective. CHO-LacZ extract had a partial inhibitory effect on the NIH 3T3 cells, probably due to endogenous CHO proteins. This inhibitory activity was not influenced by pre-incubation with antibodies.

(c) For DRG neurite outgrowth assays, the same protein material as in (b) was mixed with laminin and coated. RecNogo A had a very potent inhibitory effect on neurite outgrowth of dissociated DRG in a dose-dependent manner. The activity was neutralized by mAb IN-1 (p<0.001), but not by control mAb 01. Protein material isolated from CHO-LacZ cells was not inhibitory at any of the concentrations used, nor did incubation with antibodies have any effect on neurite outgrowth. Examples for scoring are shown in (d): 1 llg recNogo A, no or short neurites (arrows) score: 2, and in (e): 1 ug CHO-LacZ, long, branched neurites (arrowheads) score: 5-6. Statistical analysis was performed with two-tailed Student's t test. Scale bar: 280 llm.

FIG. 18: Functional Analysis of Nogo Deletion Mutants. The following deletion constructs encoding fusion proteins containing fragments of Nogo or truncated portions of Nogo (as listed below) were generated as described in Section 6.2.7 hereinbelow.

Nogo-A: His-tag/T7-tag/vector/Nogo-A seq. aa1-1162 Nogo-B: His-tag/T7-tag/vector/Nogo-A seq. aa1-171+975-1162 Nogo-C: His-tag/T7-tag/Nogo-C N-terminus (11 aa)+Nogo-A seq. aa 975-1162 NiAext: His-tag/T7-tag/vector/Nogo-A seq. aa1-974/T7-tag NiR: His-tag/T7-tag/vector/Nogo-A seq. aa1-171/vector NiG: His-tag/T7-tag/Nogo-A seq. aa 172-974/His-tag EST: His-tag/T7-tag/Nogo-A seq. aa 760-1162 NiG-D1: His-tag/T7-tag/Nogo-A seq. aa 172-908/vector NiG-D2: His-tag/T7-tag/Nogo-A seq. aa 172-866/His-tag NiG-D3: His-tag/T7-tag/Nogo-A seq. aa 172-723/His-tag NiG-D4: His-tag/T7-tag/Nogo-A seq. aa 172-646/vector NiG-D5: His-tag/T7-tag/Nogo-A seq. aa 291-646/His-tag NiG-D7: His-tag/T7-tag/Nogo-A seq. aa 172-234+292-974/His-tag NiG-D8: His-tag/T7-tag/Nogo-A seq. aa 172-628 NiG-D9: His-tag/T7-tag/Nogo-A seq. aa 172-259+646-974/His-tag NiG-D 10: His-tag/T7-tag/Nogo-A seq. aa 291-974/His-tag NiG-D14: His-tag/T7-tag/Nogo-A seq. aa 172-259 NiG-D15: His-tag/T7-tag/Nogo-A seq. aa 172-189+491-974/His-tag NiG-D16: His-tag/T7-tag/Nogo-A seq. aa 172-189+619-974/His-tag NiG-D17: His-tag/T7-tag/Nogo-A seq. aa 172-189+257-974/His-tag NiG-D18: His-tag/T7-tag/Nogo-A seq. aa 172-189+261-974/His-tag NiG-D20: His-tag/T7-tag/Nogo-A seq. aa 542-722/His-tag The amino acid (aa) numbers are based on rat Nogo A amino acid sequence numbering (SEQ ID NO: 2) starting with the first methionine. The His-tag and T7-tag consist of 34 amino acids. The N- and C-terminal vector sequences are derived from the expression vector pET28.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of Nogo genes, and amino acid sequences of their encoded proteins. The invention further relates to fragments and other derivatives, and analogs, of Nogo proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. The invention provides Nogo genes and their encoded proteins of many different species. The Nogo genes of the invention include human, rat and bovine Nogo and related genes (homologs) in other species. The bovine subsequences disclosed in Spillman et al., 1998, J. Biol. Chem. 273: 19283-19293, are not claimed as part of the present invention. In specific embodiments, the Nogo genes and proteins are from vertebrates, or more particularly, mammals. In a preferred embodiment of the invention, the Nogo genes and proteins are of human origin. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided.

The Nogo gene as provided by the present invention, encompasses nucleic acid molecules encoding three isoforms of Nogo; namely Nogo A, Nogo B and Nogo C.

Reference to the gene “Nogo” shall include nucleic acid molecules encoding all three isoforms unless otherwise specified. Likewise, reference to Nogo protein shall include all three isoforms of Nogo unless otherwise specified. Nogo proteins of the invention can prevent regeneration of neurons in the spinal cord or brain (i.e. non-permissive substrate properties), inhibit dorsal root ganglia neurite outgrowth, induce dorsal root ganglia growth cone collapse, block NIH 3T3 cell spreading, block PC12 neurite outgrowth, etc.

The Nogo proteins, fragments and derivatives thereof are free of all central nervous system myelin material; in particular, they are free of all central nervous system myelin material with which the Nogo protein is naturally associated. Such material may include other CNS myelin proteins, lipids, and carbohydrates. The Nogo proteins, fragments and derivatives thereof of the invention are also preferably free of the reagents used in purification from biological specimens, such as detergents.

In a specific embodiment, the invention provides recombinant Nogo proteins, fragments and derivatives thereof as prepared by methods known in the art, such as expressing the Nogo gene in a genetically engineered cell.

The invention also relates to Nogo derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) Nogo protein. Such functional activities include but are not limited to the ability to interact (or compete for binding) with neural growth regulatory proteins, antigenicity [ability to bind (or compete with Nogo for binding) to an anti-Nogo antibody], immunogenicity (ability to generate antibody which binds to Nogo), preventing regeneration of neurons in the spinal cord or brain, conferring to a substrate the property of restricting growth, spreading, and migration of neural cells, and neoplastic cells, inhibiting dorsal root ganglia neurite outgrowth, inducing dorsal root ganglia growth cone collapse, blocking NIH 3T3 cell spreading in vitro, blocking PC12 neurite outgrowth, restricting neural plasticity, etc.

The invention further relates to fragments (and derivatives and analogs thereof) of Nogo which comprise one or more domains of the Nogo protein.

Antibodies to Nogo, its derivatives and analogs, are additionally provided.

The present invention also relates to therapeutic and diagnostic methods and compositions based on Nogo proteins and nucleic acids and anti-Nogo antibodies. The invention provides for treatment of disorders of growth regulated cells or organs by administering compounds that promote Nogo activity (e.g., Nogo proteins and functionally active analogs and derivatives (including fragments) thereof; nucleic acids encoding the Nogo proteins, analogs, or derivatives, agonists of Nogo).

The invention also provides methods of treatment of damage or disorder of the nervous system by administering compounds that antagonize, or inhibit, Nogo function (e.g., antibodies, Nogo antisense nucleic acids, Nogo antagonist derivatives).

Animal models, diagnostic methods and screening methods for predisposition to disorders are also provided by the invention.

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections which follow.

5.1 Isolation of Nogo Genes

The invention relates to the nucleotide sequences of Nogo genes or nucleic acids. In one embodiment, Nogo nucleic acids comprise the rat cDNA sequence of FIG. 2 a (SEQ ID NO: 1) identified as Nogo A as depicted in FIG. 1 b, or the coding regions thereof, or nucleotide sequences encoding a Nogo protein of 1163 amino acids in length or any functional fragment or derivative thereof (e.g., a protein having the sequence of SEQ ID NO: 2, as shown in FIG. 2 a).

In another embodiment, Nogo nucleic acids comprise the nucleotide sequence encoding Nogo B, whereas the Nogo B protein is equivalent to the amino terminal 172 amino acids fused to the carboxy terminal 188 amino acids of Nogo A, resulting in a truncated 360 amino acid protein. The transcripts for Nogo B arise as a result of alternative splicing which removes the intervening nucleotide coding sequence.

In yet another embodiment of the present invention, Nogo nucleic acids comprise the nucleotide sequences encoding Nogo C, whereas the Nogo C protein contains 11 amino acids at its amino terminus which are not present in Nogo A, and the carboxy terminal 188 amino acids of Nogo A and B. The Nogo C protein has 199 amino acids. The transcript encoding Nogo C is the result of transcription from an alternative Nogo promoter.

In yet another specific embodiment, the present invention provides bovine Nogo nucleic acid sequences (SEQ ID NO: 28).

In yet another specific embodiment, the instant invention provides the nucleotide sequences encoding human Nogo, and fragments of human Nogo proteins, including the human equivalents to rat Nogo A, Nogo B and Nogo C. The human Nogo nucleic acid sequence is elucidated using the rat Nogo A transcript as a template and splicing together human expressed sequence tags (EST) to reveal a continuous nucleotide sequence. The rat and bovine amino acid sequences of Nogo also provided information on the proper translational reading frame such that an amino acid sequence of human Nogo is deduced.

The instant invention also provides amino acid sequences of fragments of the human Nogo gene.

The invention also provides purified nucleic acids consisting of at least 8 nucleotides (i.e., a hybridizable portion) of a Nogo sequence; in other embodiments, the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 20 nucleotides, 500 nucleotides, 700 nucleotides, or 800 nucleotides of a Nogo sequence, or a full-length Nogo coding sequence. In another embodiment, the nucleic acids are smaller than 35,200 or 500 nucleotides in length.

Nucleic acids can be single or double stranded. The invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences. In specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a Nogo gene.

In a specific embodiment, a nucleic acid which is hybridizable to a Nogo nucleic acid (e.g., having sequence SEQ ID NO: 2; FIG. 2 a), or to a nucleic acid encoding a Nogo derivative, under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78: 6789-6792): Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 pg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ug/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68° C. and reexposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations as demonstrated in the example in Section 6.1.1).

In another specific embodiment, a nucleic acid which is hybridizable to a Nogo nucleic acid under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 pg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in prehybridization mixture containing 100 Fg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filters is done at 37° C. for 1 h—in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.

In another specific embodiment, a nucleic acid, which is hybridizable to a Nogo nucleic acid under conditions of moderate stringency is provided. For example, but not limited to, procedures using such conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 h at 55° C. in a solution containing 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 ug/ml denatured salmon sperm DNA.

Hybridizations are carried out in the same solution and 5-20×106 cpm 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 55° C., and then washed twice for 30 minutes at 60° C. in a solution containing 1×SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency which may be used are well-known in the art. Washing of filters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.1% SDS. Such stringency conditions are suitable for isolating nucleic acid molecules comprising Nogo gene sequences in another species, e.g., using the rat or bovine Nogo cDNA clones as probe to isolate the human Nogo cDNA.

A number of human expressed sequence tags (ESTs) reported in published nucleic acid sequence databases display a high degree of sequence identity when compared to segments of the Nogo gene sequences of the invention. The following preliminary list of human ESTs were identified and are listed by their Genbank accession numbers: AA158636 (SEQ ID NO: 35), AA333267 (SEQ ID NO: 36), AA081783 (SEQ ID NO: 37), AA167765 (SEQ ID NO: 38), AA322918 (SEQ ID NO: 39), AA092565 (SEQ ID NO: 40), AA081525 (SEQ ID NO: 41), and AA081840 (SEQ ID NO: 42) using ENTREZ Nucleotide Query. Prior to the present invention, none of the above-identified ESTs had been characterized with respect to the amino acid sequences these ESTs may encode in vivo.

Nothing was known about the function of the proteins comprising the predicted amino acid sequences of the human ESTs. Furthermore, an EST, such as AA158636, aligning with the 5′end of rat Nogo cDNA and another EST, such as AA081840, aligning with the 3′end of rat cDNA, are not overlapping and would not be perceived to be part of the same human cDNA sequence.

Based on the Nogo gene sequences of the present invention, it is believed that these human ESTs represent portions of the human Nogo gene that are expressed in the tissue from which the ESTs were obtained. Accordingly, the present invention encompasses nucleic acid molecules comprising two or more of the above-identified human ESTs. The ESTs may be expressed in the same human tissue, or in different human tissues. Preferably, the nucleic acid molecules of the invention comprise the nucleotide sequences of at least two human ESTs which are not overlapping with respect to each other, or which do not overlap a third or more human EST.

Since the above-identified human ESTs are now identified as fragments of the human Nogo gene due to the cloning of bovine and rat Nogo nucleic acids, it is contemplated that the human ESTs have similar functions relative to the other Nogo nucleic acid molecules in various methods of the invention, such as but not limited to, for example, the expression of human Nogo polypeptides, hybridization assays, and inhibition of Nogo expression as antisense nucleic acid molecules, etc.

Moreover, the present invention provides and includes the predicted amino acid sequence of the human Nogo protein, and fragments thereof. As shown in FIG. 13, the amino acid sequence of rat Nogo protein (FIG. 2 a; SEQ ID NO: 2) is aligned with the predicted amino acid sequence of human Nogo protein (FIG. 13; SEQ ID NO: 30).

Accordingly, the present invention encompasses human Nogo proteins comprising the predicted amino acid sequence of human Nogo, FIG. 13 and SEQ ID NO: 30, or a subsequence of the predicted amino acid sequence of human Nogo, consisting of at least 6 amino acid residues, or one or more of the following predicted amino acid sequences of human Nogo fragments: MEDLDQSPLVSSS (Human Nogo, corresponding to amino acids 1-13 with SEQ ID NO: 43), KIMDLKEQPGNTISAG (Human Nogo, corresponding to amino acids 187-203 with SEQ ID NO: 44), KEDEVVSSEKAKDSFNEKR (Human Nogo, corresponding to amino acids 340-358 with SEQ ID NO: 45), QESLYPAAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASVIQPSS (Human Nogo, corresponding to amino acids 570-619 with SEQ ID NO: 46). Naturally occurring human Nogo and recombinant human Nogo, and fragments thereof having an amino acid sequence substantially similar to the above-described amino acid sequences and able to be bound by an antibody directed against a Nogo protein are within the scope of the invention.

The present invention further provides nucleic acid molecules that encodes a human Nogo protein having an amino acid sequence substantially similar to the amino acid sequence as shown in FIG. 13 (FIG. 13; SEQ ID NO: 30). In specific embodiments, nucleic acid molecules encoding fragments of human Nogo protein having an amino acid sequence substantially similar to the amino acid sequence as shown in FIG. 13 (SEQ ID NO: 30) are also contemplated with the proviso that such nucleic acid molecules do not comprise the nucleotide sequence of the above-identified human ESTs.

An amino acid sequence is deemed to be substantially similar to the predicted amino acid sequence of human Nogo protein when more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the amino acid residues in the two molecules are identical when a computer algorithm is used in which the alignment is done by a computer homology program known in the art, for example a BLAST computer searching (Altschul et al., 1994, Nature Genet. 6: 119-129) is used.

By way of example and not limitation, useful computer homology programs include the following: Basic Local Alignment Search Tool (BLAST) (www. ncbi. nlm. nih. gov) (Altschul et al., 1990, J. of Molec. Biol., 215: 403-410, “The BLAST Algorithm; Altschul et al., 1997, Nuc. Acids Res. 25: 3389-3402) a heuristic search algorithm tailored to searching for sequence similarity which ascribes significance using the statistical methods of Karlin and Altschul 1990, Proc. Nat'l Acad. Sci. USA, 87: 2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90: 5873-77. Five specific BLAST programs perform the following tasks: 1) The BLASTP program compares an amino acid query sequence against a protein sequence database.

2) The BLASTN program compares a nucleotide query sequence against a nucleotide sequence database.

3) The BLASTX program compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.

4) The TBLASTN program compares a protein query sequence against a nucleotide sequence database translated in all six reading frames (both strands).

5) The TBLASTX program compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

As will be understood by those skilled in the art, the TBLASTN program is particularly useful to identify nucleic acids with a desired percent identity and the BLASTP program is particularly useful to identify amino acid sequences with a desired percent identity.

Smith-Waterman (database: European Bioinformatics Institute wwwz.ebi.ac.uk/bicsw/) (Smith-Waterman, 1981, J. of Molec. Biol., 147: 195-197) is a mathematically rigorous algorithm for sequence alignments.

FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA, 85: 2444-2448) is a heuristic approximation to the Smith-Waterman algorithm. For a general discussion of the procedure and benefits of the BLAST, Smith-Waterman and FASTA algorithms, see Nicholas et al., 1998, “A Tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein.

The uses of the predicted amino acid sequences of human Nogo, or the nucleotide sequences of human ESTs, including degenerate sequences encoding the predicted amino acid sequence of human Nogo, for isolating or identifying the human Nogo gene, fragments, naturally occurring mutants and variants thereof, is within the scope of the invention. Such uses which will be known to one of skill in the art include but are not limited to using the information to prepare nucleic acid probes for DNA library screening, DNA amplification, genetic screening of the human population, and to prepare synthetic peptides for making antibodies. Detailed description of some of such uses are provided herein in later sections.

Nucleic acids encoding derivatives and analogs of Nogo proteins, and Nogo antisense nucleic acids are additionally provided. As is readily apparent, as used herein, a “nucleic acid encoding a fragment or portion of a Nogo protein” shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the Nogo protein and not the other contiguous portions of the Nogo protein as a continuous sequence. In this context, a portion means one or more amino acids.

Fragments of Nogo nucleic acids comprising regions conserved between (with homology to) other Nogo nucleic acids, of the same or different species, are also provided.

Nucleic acids encoding one or more Nogo domains are provided in FIG. 2 a, for example, the conserved carboxy terminal domain of rat Nogo, which has about 180 amino acids, and is encoded by the last 540 nucleotides of the coding sequence prior to the stop codon. The nucleotide and amino acid sequences of two hydrophobic domains within the conserved carboxy terminus domain, i.e., from amino acids 988 to 1023, and from amino acids 1090 to 1125, in rat Nogo A, are also provided. The nucleotide and amino acid sequences of the amino terminal acidic domain of rat Nogo A, from residues 31 to 58, are also provided.

To perform functional analysis of various regions of Nogo, a series of deletions in the Nogo gene has been generated and cloned into an expression vector by recombinant DNA techniques and expressed as a fusion protein. Nucleic acids that encode a fragment of a Nogo protein are provided, e.g., nucleic acids that encode amino acid residues 1-171, 172-974, 259-542, 542-722, 172-259, 722-974, or 975-1162 of SEQ ID NO: 2, or combinations thereof; and nucleic acids that encode amino acid residues 1-131, 132-939, 206-501, 501-680, 132-206, 680-939, and 940-1127 of SEQ ID NO: 30, or combinations thereof. Some of the deletion constructs comprises truncated portions of Nogo and additional nucleotide sequences encoding a hexahistidine tag and/or a T7-tag. Nucleic acids encoding truncated Nogo proteins that lacks amino acid residues 172-259, amino acid residues 974-1162, or amino acid residues 172-259 and 974-1162, of SEQ ID NO: 2 but otherwise comprises the remainder of SEQ ID NO: 2; or amino acid residues 132-206, amino acid residues 939-1127, or amino acid residues 132-206 and 939-1127, of SEQ ID NO: 30 but otherwise comprises the remainder of SEQ ID NO: 30, are provided. The structure of exemplary deletion constructs are shown in FIG. 18. The deletion constructs produce fragments or truncated portion (s) of Nogo when introduced into a cell. The biological activities of these mutants were tested in various functional assays as described in Table 2 in Section 6.2.7.—Specific embodiments for the cloning of a Nogo gene, presented as a particular example but not by way of limitation, follows: For expression cloning (a technique commonly known in the art), an expression library is constructed by methods known in the art. For example, mRNA (e.g., human) is isolated, cDNA is made and ligated into an expression vector (e.g., a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed Nogo product. In one embodiment, anti-Nogo antibodies can be used for selection.

In another embodiment, polymerase chain reaction (PCR) is used to amplify the desired sequence in a genomic or cDNA library, prior to selection. Oligonucleotide primers representing known Nogo sequences can be used as primers in PCR. In a preferred aspect, the oligonucleotide primers represent at least part of the Nogo conserved segments of strong homology between Nogo of different species. The synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). The DNA being amplified can include mRNA or cDNA or genomic DNA from any eukaryotic species. One can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleotide sequence similarity between the known Nogo nucleotide sequence and the nucleic acid homolog being isolated. For cross species hybridization, low stringency conditions are preferred. For same species hybridization, moderately stringent conditions are preferred.

After successful amplification of a segment of a Nogo homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In this fashion, additional genes encoding Nogo proteins and Nogo analogs may be identified.

The above-methods are not meant to limit the following general description of methods by which clones of Nogo may be obtained.

Any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of the Nogo gene. The nucleic acid sequences encoding Nogo can be isolated from vertebrate, mammalian, human, porcine, murine, bovine, feline, avian, equine, canine, as well as additional primate sources, insects, etc. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA “library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell. (See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U. K. Vol. I, II.) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a Nogo (of any species) gene or its specific RNA, or a fragment thereof (see Section 6.1.1), is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196: 180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72: 3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene.

Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, post-translational modifications, acidic or basic properties or antigenic properties as known for Nogo. Antibodies to Nogo are available, such as IN-1 and IN-2 (U.S. Pat. No. 5,684,133), AS Bruna and AS 472.

Preparation of AS Bruna and AS 472 are described in Section 6.1. 7. The Nogo protein may be identified by binding of labeled antibody to the putatively Nogo synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure or by western blotting of purified or whole cell extracts.

The Nogo gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified Nogo DNA of another species (e.g., mouse, human).

Immunoprecipitation analysis or functional assays (e.g., aggregation ability in vitro; binding to receptor; see infra) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against Nogo protein. A radiolabeled Nogo cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabeled mRNA or cDNA may then be used as a probe to identify the Nogo DNA fragments from among other genomic DNA fragments.

Alternatives to isolating the Nogo genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes the Nogo protein. For example, RNA for cDNA cloning of the Nogo gene can be isolated from cells which express Nogo. Other methods are possible and within the scope of the invention.

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene). In a specific example, Nogo is cloned into pcDNA3 with epitope tags for simplified protein expression analysis (Section 6.1.10).

The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified.

Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and Nogo gene may be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.—In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a “shot gun” approach. Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated Nogo gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

The Nogo sequences provided by the instant invention include those nucleotide sequences encoding substantially the same amino acid sequences as found in native Nogo proteins, and those encoded amino acid sequences with functionally equivalent amino acids, as well as those encoding other Nogo derivatives or analogs, as described in Sections 6.2.1 and 6.2.2 infra for Nogo derivatives and analogs.

5.2 Expression of the Nogo Genes

The nucleotide sequence coding for a Nogo protein or a functionally active analog or fragment or other derivative thereof (see FIGS. 1 b and 2 a; Sections 6.2.1 and 6.2.2), can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals can also be supplied by the native Nogo gene and/or its flanking regions. The coding sequence can also be tagged with a sequence that codes for a well described antigen or biological molecule that has known binding properties to a binding partner (e.g. myc epitope tag, histidine tag, T7 epitope tag etc., see Section 6.2.6 and FIG. 11 a-11 c). This additional sequence can then be exploited to purify the Nogo protein, protein fragment, or derivative using the interaction of the binding group with its corresponding partner, which is attached to a solid matrix.

A variety of host-vector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccina virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. In specific embodiments, the human Nogo gene is expressed, or a sequence encoding a functionally active portion of human Nogo, as a specific example, either Nogo A, Nogo B or Nogo C is expressed (FIG. 1 b). In yet another embodiment, a fragment of Nogo comprising a domain of the Nogo protein is expressed.

As used herein, a cell is “transformed” with a nucleic acid, when such cell contains a nucleic acid not natively present in the cell, after introduction of the nucleic acid into the cell or its ancestor, e.g., by transfection, electroporation, transduction, etc.

Nucleotide sequences encoding fragments of human Nogo A comprising an amino acid sequence selected from the group consisting of amino acid residues 1-131, 132-939, 680-939, and 940-1127 of SEQ ID NO: 30 are also provided.

Nucleotide sequences that encodes truncated portions of human Nogo A are also provided; the truncated proteins lack amino acid residues 132-206, amino acid residues 939-1127, or amino acid residues 132-206 and 939-1127, of SEQ ID NO: 30 but otherwise comprises the remainder of SEQ ID NO: 30.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences.

These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequence encoding a Nogo protein or peptide fragment may be regulated by a second nucleic acid sequence so that the Nogo protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a Nogo protein may be controlled by any promoter/enhancer element known in the art. An exemplary embodiment is to use one of Nogo's natural promoters, either P1 or P2, discussed in Section 6.2.1. A non-native promoter may also be used. Promoters which may be used to control Nogo expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22: 787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors such as the p-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75: 3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303: 209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9: 2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella—et al., 1984, Nature 310: 115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcool dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50 : 399-409; MacDonald, 1987, Hepatology 7: 425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48: 703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314: 283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

In a specific embodiment, a vector is used that comprises a promoter operably linked to a Nogo-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).

In a specific embodiment, an expression construct is made by subcloning a Nogo, coding sequence into the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, 1988, Gene 7: 31-40).

This allows for the expression of the Nogo protein product from the subclone in the correct reading frame.

Expression vectors containing Nogo gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of “marker” gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a Nogo gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted Nogo gene. In the second approach, the recombinant vector/host system can be identified and selected based' upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a Nogo gene in the vector. For example, if the Nogo gene is inserted within the marker gene sequence of the vector, recombinants containing the Nogo insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the Nogo product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the Nogo protein in vitro assay systems, e.g., binding with anti-Nogo antibody.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered Nogo protein may be controlled.

Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure “native” glycosylation of a heterologous protein.

Furthermore, different vector/host expression systems may effect processing reactions to different extents.

In other specific embodiments, the Nogo protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.

Both cDNA and genomic sequences can be cloned and expressed.

5.3 Identification and Purification of the Nogo Gene Products

In particular aspects, the invention provides amino acid sequences of Nogo, preferably human Nogo, and fragments and derivatives thereof which comprise an antigenic determinant (i.e., can be recognized by an antibody) or which are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing. “Functionally active” Nogo material as used herein refers to that material displaying one or more known functional activities associated with a full-length (wild-type) Nogo A protein, e.g., non-permissive substrate properties, dorsal root ganglia growth cone collapse, NIH 3T3 spreading inhibition, neurite outgrowth inhibition, binding to a Nogo substrate or Nogo binding partner, antigenicity (binding to an anti-Nogo antibody), immunogenicity, etc.

In specific embodiments, the invention provides fragments of a Nogo protein consisting of at least 6 amino acids, 10 amino acids, 17 amino acids, 50 amino acids, 100 amino acids or of at least 220 amino acids. In other embodiments, the proteins comprise or consist essentially of the highly conserved Nogo carboxy terminal domain (carboxy terminal 188 amino acids of Nogo A). Fragments, or proteins comprising fragments, lacking the conserved carboxy terminal domain, or the hydrophobic carboxy terminal stretches, or the amino terminal acidic domain, or the amino terminal poly-proline region or any combination thereof, of a Nogo protein are also provided. Nucleic acids encoding the foregoing are provided.

Once a recombinant which expresses the Nogo gene sequence is identified, the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, etc.

Once the Nogo protein is identified, it may be isolated and purified by standard methods including chroma. 

1. A binding molecule, which immunospecifically binds to a fragment of a protein, wherein said protein has an amino acid sequence of at least 90% identity to the amino acid sequence of SEQ ID NO:29, wherein said fragment consists of at least 10 amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:45, amino acids 183-989 of SEQ ID NO:29, amino acids residues 132-939 of SEQ ID NO:29, or amino acids 206-501 of SEQ ID NO:29, wherein said fragment inhibits spreading of PC12 cells.
 2. The binding molecule of claim 1, wherein said fragment consists of at least 17 amino acids of an amino acid sequence selected from the group consisting of: SEQ ID NO:45, amino acid residues 183-989 of SEQ ID NO:29, amino acids residues 132-939 of SEQ ID NO:29, or amino acids 206-501 of SEQ ID NO:29.
 3. The binding molecule of claim 2, wherein said fragment consists of has an amino acid sequence selected from the group consisting of: SEQ ID NO:45, amino acids 183-989 of SEQ ID NO:29, amino acid residues 183-731 of SEQ ID NO:29, amino acids residues 132-939 of SEQ ID NO:29, or amino acids 206-501 of SEQ ID NO:29.
 4. The binding molecule of any one of claims 1-3, wherein said fragment is a synthetic peptide.
 5. The binding molecule of any one of claims 1-3, which is a monoclonal antibody.
 6. The binding molecule of any one of claims 1-3, which is a polyclonal antibody.
 7. The binding molecule of claim 5, wherein said antibody is selected from the group consisting of: a human antibody, a chimeric antibody, a single chain antibody, a Fab-fragment, a Fab' fragment, an Fv-fragment, or an F(ab')₂ fragment of an antibody.
 8. The binding molecule of claim 6, wherein said antibody is a human antibody.
 9. A composition comprising a binding molecule of any one of claims 1-3, and a carrier.
 10. A pharmaceutical composition comprising a binding molecule of any one of claims 1-3, and a pharmaceutically acceptable carrier.
 11. A method for treating damage to the central nervous system comprising administering to a subject in need thereof, a therapeutically effective amount of the binding molecule of any one of claims 1-3.
 12. A method for promoting plasticity of the central nervous system comprising administering to a subject in need thereof, a therapeutically effective amount of the binding molecule of any one of claims 1-3.
 13. A method for inducing regeneration of sprouting neurons comprising administering to a subject in need thereof, a therapeutically effective amount of the binding molecule of any one of claims 1-3.
 14. The method of claim 11, wherein said subject is human.
 15. The method of claim 12, wherein said subject is human.
 16. The method of claim 13, wherein said subject is human. 